The integrin-linked kinase (ILK) is critical for anchorage-dependent cell growth and survival, cell cycle progression, epithelial to mesenchymal transition, cell motility, contractility and early development. ILK is also required for cardiac, vascular, brain, kidney, muscle, skin, platelet, chondrocyte and T cell function and plays important roles in tumor angiogenesis. There are multiple signaling pathways downstream of integrins but many of these pathways require the formation of a heterotrimeric complex between ILK, PINCH and parvin (IPP). This IPP complex serves as a hub in integrin-actin and integrin-signaling networks, and in mammalian systems IPP complex formation precedes and is required for its correct targeting to adhesions. There are currently significant deficiencies in our understanding of how the IPP complex forms, how it interacts with integrins, and whether it is enzymatically competent. We aim to improve the understanding of integrin signaling by the IPP complex using a structure-directed functional approach and to resolve the key functional question of whether ILK is catalytically active. In Aim 1 we will determine crystal structures and conduct binding studies to provide a comprehensive molecular description of the interaction between ILK and PINCH. These biophysical studies will allow us to rationally investigate the cellular effects of targeted interruption of this interaction. In Aim 2 we will provide structural, enzymatic and functional analyses of the ILK kinase domain. The catalytic competence of this pseudokinase remains the subject of much controversy, but is reported to be critical for IPP-mediated integrin signaling. We will solve crystal structures of the ILK kinase domain and assess its catalytic activity to provide structural evidence for the molecular basis of ILK catalytic competence. Potential substrate specificities will be studied as will the role of intramolecular interactions on kinase activity. Using structure-guided site-directed mutagenesis we will investigate the functional role of ILK catalytic activity in cells. In Aim 3 we will determine the molecular basis for ILK interactions with parvin and whether ILK directly interacts with integrin 2 subunit cytoplasmic tails. If ILK binds integrin 2 tails we will determine the structural basis for this interaction and investigate the functional effects of its targeted disruption. In Aim 4 we will determine the molecular architecture of the complete IPP complex. This long-term goal will provide a structural description of how the IPP complex forms and will allow targeted functional analysis of its cellular role. The studies proposed will provide answers to some of the critical unresolved questions regarding integrin signaling by the IPP complex. They may also facilitate the design of targeted anti-ILK or anti-IPP therapeutics relevant to the treatment of cancer, cardiovascular and inflammatory and kidney diseases. Our results will significantly enhance the molecular, enzymatic and functional understanding of a critical integrin signaling complex. PUBLIC HEALTH RELEVANCE: The project aims to enhance our molecular and functional understanding of integrin-linked kinase-PINCH- parvin (IPP) complex. This complex is essential for embryonic development, tissue maintenance and repair, host defense and hemostasis. We will determine crystal structures, conduct functional studies and describe the effects of mutations in cells. The proposed studies will help us understand how the IPP complex mediates its critical cellular roles.